PowerPoint® Lecture

Presentations prepared by
Mindy Miller-Kittrell,
North Carolina
State University

CHAPTER 4
Microscopy, Staining,
and Classification

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 Microscopy and Staining: Overview

PLAY Microscopy and Staining: Overview

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© 2014 Pearson Education, Inc.
 Microscopy

• General Principles of Microscopy

• Wavelength of radiation

• Magnification

• Resolution

• Contrast

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 Figure 4.1 The electromagnetic spectrum.

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 Figure 4.2 Light refraction and image magnification by a convex glass lens.

Light

Air Glass

Focal point

Inverted,
Specimen Convex
lens reversed, and
Enlarged
image
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 Figure 4.3 The limits of resolution (and some representative objects within those ranges) of the human eye and of
various types of microscopes.

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 Microscopy

• General Principles of Microscopy

• Contrast
• Differences in intensity between two objects, or an object
and its background
• Important in determining resolution

• Staining increases contrast

• Use of light that is in phase increases contrast

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 Microscopy

• Light Microscopy
• Bright-field microscopes
• Simple

• Contain a single magnifying lens

• Similar to magnifying glass

• Leeuwenhoek used simple microscope to observe

microorganisms

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 Microscopy

• Light Microscopy
• Bright-field microscopes

• Compound

• Series of lenses for magnification

• Light passes through specimen into objective lens

• Oil immersion lens increases resolution

• Have one or two ocular lenses

• Total magnification = magnification of objective lens X

magnification of ocular lens
• Most have condenser lens (direct light through specimen)
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 Figure 4.4 A bright-field, compound light microscope.

Line of vision
Ocular lens
Remagnifies the image formed
by
the objective lens
Body Ocular lens
Transmits the image from the
objective lens to the ocular lens Path of light
using prisms
Arm Prism

Objective lenses Body

Primary lenses that
magnify the specimen Objective
Stage lenses
Holds the microscope
slide in position Specimen
Condenser
Focuses light Condenser
through specimen lenses
Diaphragm
Controls the amount of Illuminator
light entering the condenser
Illuminator
Light source

Coarse focusing knob

Moves the stage up and
down to focus the image
Fine focusing knob

Base

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 Figure 4.5 The effect of immersion oil on resolution.

Microscope Microscope
objective Lenses objective

Refracted light More light

rays lost to lens enters lens
Immersion oil

Glass cover slip Glass cover slip

Slide Slide

Specimen Light source Light source

Without immersion oil With immersion oil

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 Microscopy

• Light Microscopy
• Dark-field microscopes
• Best for observing pale objects

• Only light rays scattered by specimen enter objective lens

• Specimen appears light against dark background

• Increases contrast and enables observation of more details

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 Figure 4.6 The light path in a dark-field microscope.
Objective

Light refracted
by specimen

Light unrefracted
by specimen

Specimen

Condenser

Dark-field stop Dark-field stop

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 Microscopy

• Light Microscopy
• Phase microscopes
• Used to examine living organisms or specimens that would
be damaged/altered by attaching them to slides or staining
• Light rays in phase produce brighter image, while light rays
out of phase produce darker image
• Contrast is created because light waves are out of phase
• Two types
• Phase-contrast microscope
• Differential interference contrast microscope
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 Figure 4.7 Principles of phase microscopy.

Rays in phase Rays out of phase

Phase plate

Bacterium Ray deviated by Deviated ray

specimen is 1/4 is now 1/2
wavelength out Wavelength
of phase. out of phase.

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 Figure 4.8 Four kinds of light microscopy.
Nucleus

Bacterium

Bright field Dark field

Phase contrast Nomarski

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 Microscopy

• Light Microscopy
• Fluorescent microscopes
• Direct UV light source at specimen
• Specimen radiates energy back as a longer, visible
wavelength
• UV light increases resolution and contrast
• Some cells are naturally fluorescent; others must be
stained
• Used in immunofluorescence to identify pathogens and to
make visible a variety of proteins
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 Figure 4.9 Fluorescence microscopy.

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 Figure 4.10 Immunofluorescence.

Antibodies Fluorescent dye

Antibodies
carrying dye
Bacterium
Cell-surface
antigens

Bacterial cell with

bound antibodies
carrying dye

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 Microscopy

• Light Microscopy
• Confocal microscopes
• Use fluorescent dyes

• Use UV lasers to illuminate fluorescent chemicals in a

single plane
• Resolution increased because emitted light passes
through pinhole aperture
• Computer constructs 3-D image from digitized images

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 Light Microscopy

PLAY Light Microscopy

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 Microscopy

• Electron Microscopy
• Light microscopes cannot resolve structures closer than 200 nm
• Electron microscopes have greater resolving power and
magnification
• Magnifies objects 10,000X to 100,000X
• Detailed views of bacteria, viruses, internal cellular structures,
molecules, and large atoms
• Two types
• Transmission electron microscopes
• Scanning electron microscopes
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 Figure 4.11 A transmission electron microscope (TEM).

Light microscope Column of transmission

(upside down) electron microscope

Lamp Electron gun

Condenser
lens
Specimen Specimen

Objective lens Objective lens

(magnet)

Eyepiece Projector lens

(magnet)

Final image Final image on

seen by eye fluorescent screen

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 Figure 4.12 Scanning electron microscope (SEM).

Electron gun

Magnetic Beam
lenses deflector coil

Scanning
Primary circuit
electrons

Secondary
electrons
Photo-
Specimen multiplier Monitor
Specimen Detector
holder

Vacuum
system
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 Figure 4.13 SEM images.

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 Electron Microscopy

PLAY Electron Microscopy

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 Microscopy

• Probe Microscopy
• Magnifies more than 100,000,000 times

• Two types
• Scanning tunneling microscopes

• Atomic force microscopes

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 Figure 4.14 Probe microscopy.
DNA Enzyme

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© 2014 Pearson Education, Inc.
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 Staining

• Most microorganisms are difficult to view by bright-

field microscopy
• Coloring specimen with stain increases contrast
and resolution
• Specimens must be prepared for staining

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 Figure 4.15 Preparing a specimen for staining.

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 Staining

• Principles of Staining
• Dyes used as stains are usually salts

• Chromophore is the colored portion of the dye

• Acidic dyes stain alkaline structures

• Basic dyes stain acidic structures

• More common since most cells are negatively charged

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 Staining

• Simple stains
• Differential stains
• Gram stain
• Acid-fast stain
• Endospore stain

• Special stains
• Negative (capsule) stain
• Flagellar stain

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 Figure 4.16 Simple stains.

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 Figure 4.17 The Gram staining procedure.

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 Figure 4.18 Ziehl-Neelsen acid-fast stain.

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 Figure 4.19 Schaeffer-Fulton endospore stain of Bacillus anthracis.

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 Staining

• Differential Stains
• Histological stains
• Two common stains used for histological specimens

• Gomori methenamine silver (GMS) stain

• Hematoxylin and eosin (HE) stain

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 Figure 4.20 Negative (capsule) stain of Klebsiella pneumoniae.

Bacterium

Capsule

Background
stain

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 Figure 4.21 Flagellar stain of Proteus vulgaris.

Flagella

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 Staining

• Staining for Electron Microscopy

• Chemicals containing heavy metals used for
transmission electron microscopy
• Stains may bind molecules in specimens or the
background

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 Staining

PLAY Staining

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 Classification and Identification of
Microorganisms
• Taxonomy consists of classification,
nomenclature, and identification
• Organize large amounts of information about
organisms
• Make predictions based on knowledge of similar
organisms

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© 2014 Pearson Education, Inc.
 Classification and Identification of
Microorganisms
• Linnaeus and Taxonomic Categories
• Linnaeus
• His system classified organisms based on characteristics
in common
• Grouped organisms that can successfully interbreed into
categories called species
• Used binomial nomenclature

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 Figure 4.22 Levels in a Linnaean taxonomic scheme.

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 Classification and Identification of
Microorganisms
• Linnaeus and Taxonomic Categories
• Linnaeus proposed only two kingdoms
• Later taxonomic approach based on five kingdoms
• Animalia, Plantae, Fungi, Protista, and Prokaryotae
• Linnaeus's goal was classifying organisms to catalog them
• Modern goal is understanding relationships among
organisms
• Goal of modern taxonomy is to reflect phylogenetic hierarchy
• Greater emphasis on comparisons of organisms' genetic
material led to proposal to add domain
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 Classification and Identification of
Microorganisms
• Domains
• Carl Woese compared nucleotide sequences of rRNA
subunits
• Proposal of three domains as determined by ribosomal
nucleotide sequences
• Eukarya, Bacteria, and Archaea

• Cells in the three domains also differ with respect to

many other characteristics

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 Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Physical characteristics

• Biochemical tests

• Serological tests

• Phage typing

• Analysis of nucleic acids

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 Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Physical characteristics
• Can often be used to identify microorganisms

• Protozoa, fungi, algae, and parasitic worms can often

be identified based only on their morphology
• Some bacterial colonies have distinct appearance
used for identification

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 Figure 4.23 Two biochemical tests for identifying bacteria.
Gas bubble Inverted tubes to trap gas

Hydrogen No
sulfide hydrogen
Acid with gas Acid with no gas Inert produced sulfide

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 Figure 4.24 One tool for the rapid identification of bacteria, the automated MicroScan system.

Wells

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 Figure 4.25 An agglutination test, one type of serological test.

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 Figure 4.26 Phage typing.
Bacterial lawn

Plaques

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 Classification and Identification of
Microorganisms
• Taxonomic and Identifying Characteristics
• Analysis of nucleic acids
• Nucleic acid sequence can be used to classify and
identify microbes
• Prokaryotic taxonomy now includes the G + C content of
an organism's DNA

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 Classification and Identification of
Microorganisms
• Taxonomic Keys
• Dichotomous keys
• Series of paired statements where only one of two "either/
or" choices applies to any particular organism

• Key directs user to another pair of statements, or

provides name of organism

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 Figure 4.27 Use of a dichotomous taxonomic key.

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 Dichotomous Keys: Overview

PLAY Dichotomous Keys: Overview

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